Immunological activity for a peptide of the limulus anti-LPS factor

ABSTRACT

The present invention relates a novel immunological effect of one peptide from Limulus anti-LPS factor protein. The immunological effect are, (1) Induce an antiviral state in both the Hep-2 and MDBK cell lines, (2) Supernatant from human mononuclear cells stimulated with peptide Limulus anti-LPS factor (LALF) is able to induce antiviral effect on Hep-2 cell line by mean of IFN-γ (3) LALF peptide is able to modulate the immune response in vitro and in vivo. The present invention can be used in therapeutic and/or prophylactic treatment regimens of humans and animals to enhance their immune responses, without stimulating the production of certain biochemical mediators (e.g., TNF-α) that can cause detrimental effects, such as fever and inflammation.

This application is a divisional application of U.S. Ser. No. 09/160,309filed Sep. 25, 1998, now U.S. Pat. No. 6,191,114.

Despite aggressive management, septic shock arising from Gram-negativesepsis continues to be a leading cause of death in both surgical andmedical patients. Death in such patients usually results fromcardiovascular collapse and/or multiple organ system failure. One of themain components of Gram-negative bacterial thought to play an integralrole in causing septic shock is an outer wall constituent, endotoxin(LPS).

Endotoxins are high molecular weight complexes, associated with theouter membrane of Gram-negative bacteria that produce pyrogenicreactions upon intravenous administration. Endotoxin is shed from livingbacteria and is also released into the environment when bacteria die anddecompose.

Bacterial endotoxin is a complex consisting of lipid, carbohydrate andprotein. It is characterized by an overall negative charge, heatstability and high molecular weight. Highly purified endotoxin does notcontain protein, and is a lipopolysaccharide (LPS).

Bacterial endotoxins are known to have profound biological effects inanimals and humans, and to cause severe medical problems when present.Symptoms include induction of high fever, activation of complement andcytokine cascade and hypotension. It is critical to avoid endotoxincontamination in any pharmaceutical product or medical device, whichcome into contact with body fluids. High endotoxin levels in sera due tobacterial diseases, such as septicemia, are not easily treated.Antibiotic treatment of the infection only kills the bacteria, leavingthe endotoxin from their cell walls free to cause fever.

LPS is an important mediator in the pathogenesis of septic shock and isone of the major causes of death in intensive-care units in the UnitedStates. It has been observed that exposure to LPS during sepsisstimulates an immune response in monocytes and macrophages that resultsin a toxic cascade resulting in the production of TNF-α and otherproinflammatory cytokines. Morrison and Ulevitch, Am. J. Pathol., 93:527(1978). Endothelial damage in sepsis probably results from persistentand repetitive inflammatory insults. Bone, Annals Int. Med. 115:457(1991).

Several proteins have been investigated for their ability to bind andneutralize LPS and the potential use in the septic shock treatment.Among the most extensively studied of the LPS-binding proteins isbactericidal/permeability-increasing protein (BPI), a basic proteinfound in the azurophilic granules of polymorphonuclear leukocytes. TheBPI protein from human PMNs has potent bactericidal activity against abroad spectrum of Gram-negative bacteria. This antibacterial activityappears to be associated with the amino terminal region (amino acidsresidues 1-199) of the isolated human BPI protein. Recently it has beenshown that the N-terminal fragment of BPI (rBPI₂₅) neutralizes endotoxinactivities and inhibits LPS-induced events in neutrophiles andmacrophages. Helene et al., Infection and Immunity. 62:1185 (1994). Inaddition to its bactericidal effects, BPI has been shown to neutralizethe toxic and cytokine-inducing effects of LPS to which it binds. XomaCorporation is building a portfolio of therapeutic products based on BPIprotein. The company's lead BPI-derived product, Neuprex, is in clinicalefficacy trials for four indications: [1] Meningococcemia, [2]Hemorrhagic trauma, [3] Partial hepatectomy, [4] Severe intro-abdominalinfections. XOMA Corporation Jul. 30, 1996.

Lipopolysaccharide binding protein (LBP) is a 60 kD glycoproteinsynthesized in the liver, which shows significant structural homologywith BPI. Shumann et al. Disclose the amino acid sequences and encodingcDNA of both human and rabbit. Like BPI, LBP has a binding site forlipid A and binds to the LPS from rough and smooth form bacterial.Unlike BPI, LBP does not possess significant bactericidal activity, andit enhances (rather than inhibits) LPS-induced TNF production. Schumannet al., Science, 249:1429 (1990).

One of the normal host effector mechanisms for clearance of bacteriainvolves the binding to and subsequent phagocytosis by neutrophils andmonocytes. As part of this process, bacteria are exposed to bactericidaland bacteriostatic factor, including oxygen radicals, lysosomal enzymes,lactoferrin and various cationic proteins. LBP opsonizes LPS-bearingparticles and intact Gram-negative bacteria, mediating attachment ofthese LPB-coated particles to macrophages. Wright et al., J. Exp. Med.170: 1231 (1989). The attachment appears to be through the CD14 receptorof monocytes, which bind complexes of LPS and LBP. Wright et al. Science249: 1431 (1990). Interaction of CD14, which is present on the surfaceof polymorphonuclear leukocytes as well as monocytes, with LPS in thepresence of LBP has been shown to increase the adhesive activity ofneutrophils. Wright et al., J. Exp. Med. 173: 281 (1991), Worthen etal., J. Clin. Invest. 90: 2526 (1992). Thus, while BPI has been shown tobe cytotoxic to bacteria and to inhibit proinflammatory cytokineproduction stimulated by bacteria, LBP promotes bacterial binding to andactivation of monocytes through a CD14-dependent mechanism. Novelbiologically active lipopolysaccharide binding protein (LBP) derivativesincluding LBP derivative hybrid proteins which are characterized by theability to bind to and neutralize LPS and which lack the CD14-mediatedimmunostimulatory properties of holo-LBP. Gazzano-Santoro, WO 95/00641.Moreover, peptides corresponding to residues 91-108 of LBP protein wereidentified that specifically bound the lipid A with high affinity. Thepeptides inhibited binding of LPS to LBP, inhibited the chromogenicLimulus amebocyte lysate reaction, and blocked release of TNF followingLPS challenge both in vitro and in vivo, Taylor²⁴ et al., J. of Biol.Chem. 270: 17934, (19951.

Another LPS-binding proteins capable to bind and neutralize theendotoxin have been isolated from a horseshoe crab such as Limuluspolyphemus and Tachypleus antilipopolysaccharide factor (TALF) isolatedfrom Tachypleus tridentatus, Kloczewiak²⁹ et al., J. Infect. Dis. 170:1490-7 (1994). . The cells from their hemolymph (amebocytes) undergo acomplex series of biochemical reactions resulting in clot formation,analogous to mammalian blood coagulation. This phenomenon has beenexploited in the form of bioassays sensitive to very low endotoxinlevels.

Currently, a bioassay of this type is the method of choice formonitoring pharmaceutical manufacturing and is termed Limulus AmebocyteLysate (LAL). Wainwright et al., WO 92/20715 relates the invention tothe pharmaceutical utility of the endotoxin binding/neutralizing proteinand disclose the use of the endotoxin binding/neutralizing protein foran endotoxin assay. It is yet another object of the invention to providepharmaceutical compositions capable of binding and neutralizingendotoxin in vivo and containing therein an endotoxinbinding/neutralizing protein corresponding at least to part of theendotoxin binding and neutralizing domain of the endotoxinbinding/neutralizing protein isolated from a horseshoe crab inaccordance with the invention.

Limulus anti-LPS factor (LALF) have been investigated for use in sepsis.Warren et al., Infect.Immun. 60: 2506-2513 (1992) and Garcia²⁵ et al.,Crit. Care. Med. 22: 1211 [1994]. This protein is almost certain tosuffer the disadvantages associated with other foreign proteins forhuman therapy, it is immunogenic and has only a shart half-life incirculation. These factors will reduce its clinical potential. None ofthese substances have been proven to be effective for the treatment ofthe serious conditions associated with Gram-negative infection inhumans.

The solution to the above technical problem was achieved by providingsubstances, which relate to peptides, which bind tightly to LPS, andtherefore have utility in the diagnosis and treatment of Gram-negativeand other septic conditions. Battafaraono et al., synthesized threepeptides from BPI, LALF, LBP (each 27 amino acid in length) of theproposed LPS-binding motif for these proteins. All small peptidesderived from BPI, LALF and LBP retained significantendotoxin-neutralizing and bactericidal activity against many differentgram-negative bacterial in vitro Battafaraono²⁶ et al., Surgery 118:318-24 [1995]. More recently, Fletcher et al., designed a novelpeptide-IgG conjugate, CAP-18 (106-138)-IgG, it binds and neutralizesendotoxin and kills gram-negative bacteria. Fletcher²⁷ et al., J.Infect. Dis. 175: 621-32 [1997].

Hoess et al., WO 95/05393 relates the invention to substances which bindwith high affinity to endotoxin (lipopolysaccharide [LPS]), and whichare useful for the prevention or treatment of, for example,Gram-negative and Gram-positive bacterial sepsis, and for the treatmentof bacterial and fungal infections as well as for neutralizing effectsassociated with heparin. The substances are LPS-binding peptidescomprising an LPS-binding domain. A peptide comprising the aminoacids₃₁₋₅₂ from Limulus anti-LPS factor (LALF) was disclosed.

The crystal structure of LALF reveals a simple tertiary fold, which hasa striking shape and amphipathicity. A surface-extended loop in the LALFstructure (loop of LALF or LALF-loop) has similar features to polymyxinB by being positively charged and amphipathic and having several exposedhydrophobic and aromatic residues. Furthermore, the loop of LALF isdistinguished by an alternating series of positively charged andhydrophobic/aromatic residues that, by virtue of the extendedβ-conformation, point in opposite directions, and a single pair ofpositive charges, that, because of the β-turn conformation, point in thesame direction and maintain the amphipathicity. The loop contains nonegatively charged amino acids. Hoess had described the minimalrequirements of rLALF for endotoxin and lipid A with linear 10-merpeptides. Cyclic peptides, however, bind lipid A and endotoxin with highaffinity, presumably by mimicking the three dimensional characteristicsof the exposed hairpin loop, Ried²⁸ et al., J. Biol. Chem. 271: 28120-7[1996]. The cyclic peptide LALF₃₆₋₄₇ is able to blocks TNF inductionafter endotoxin challenge in mice. Most recently have been describedthat Limulus antilipopolysaccharide factor (LALF) neutralize bacterialendotoxin and protect mice from LPS lethality even when LALF isadministered long after the onset of continuous endotoxemia, Roth³¹ RI.Su D H. Child A H. Wainwright N R and Levin J. Journal of InfectiousDiseases. 177 (2): 388-394, 1998.

A similar amphipathic loop exists in three other proteins which bindLPS: rabbit and human lipopolysaccharide-binding protein (LBP) and humanbactericidal/permeability-increasing protein (BPI). Inspection of theLBP and BPI sequences reveals a similar pattern of alternating residuesthat could produce an amphipathic loop.

However, these inventions do not reveal anything about the antiviraleffect and/or activation of monocytes of either the endotoxinbinding/neutralizing protein from Limulus polyphemus or the peptidecomprising the amino acids₃₁₋₅₂ (SEQ ID NO: 1) from the protein. The useor therapeutic effectiveness in sepsis and infectious diseases arerelated with the LPS-binding. Furthermore, none of the referencesdisclose the use of the peptide₃₁₋₅₂ (SEQ ID NO: 1) from Limulusanti-LPS factor for an antiviral treatment or to enhance the immuneresponse.

It is known that viruses are small infectious agents that contain onlyone type of nucleic acid in its genome (RNA or DNA), generally as a solemolecule. Viruses replicate only in living cells and in man they mayproduce different diseases.

A. Generalized diseases where the virus is disseminated throughout thebody as in small pox, yellow fever, dengue, etc.

B. Diseases of the nervous system as in poliomyelitis, asepticmeningitis caused by the intestinal viruses (polio, coxsackie), rabies,encephalitis transmitted by arthropods, etc.

C. Diseases of the skin or mucose: Herpes simplex, type I (usually ofthe mouth) and type II (usually genital).

D. Ocular diseases: Conjunctivitis due to adenovirus, conjunctivitis dueto the Newcastle virus, epidermal hemorrhagic conjunctivitis.

E. Diseases of the liver: Type A Hepatitis (infectious hepatitis), typeB hepatitis (hepatitis through the serum), etc.

Given that the viruses are necessarily intracellular parasites, theantiviral agents should be capable of selectively inhibiting the viralfunctions without damaging the host. There are several compounds thatinhibit the virus in a specific way without affecting cellularmetabolism, for example the amantadine, a synthetic amine that inhibitsthe virus A of the influenza; metisazone, an inhibitor of many poxviruses, the trifluorotimidine has been used with success in thetreatment of lesions of the cornea due to the Herpes simplex virus. Manyof these agents are acting as antimetabolites have the handicap thatthey are toxic to man.

Also tested because of their antiviral capacity and immune systemstimulating ability in animals and man are compounds such as Levamisoland Isoprinosine, these agents are not antimetabolites, but stimulatorsof cellular immunity.

The interferons, a kind of protein discovered in 1957, capable ofinhibiting viral replication, are produced by animals and man, as wellas by cultured cells, as a response to viral infection or some otherinductor. Interferon is more effective as an antiviral substance in thecells of the same species. In contrast, the interferon activity is notspecific for a certain virus. The interferon produced as a response to avirus or to another inductor, effectively inhibits the replication of awide variety of viruses. When the interferon is added to the cellsbefore the infection take place, there is a notable inhibition of viralreplication, while the cell function remain normal.

Viral infections in man may also be associated with complications duringserious surgical operations as in gastrointestinal surgery, or inpatients having a high risk of infection at the site of the operation asin cardiovascular surgery.

In aseptic surgical operations such as abdominal or cardiovascularsurgery, the so-called post-operational acute phase reaction ischaracterized by a deterioration of the phagocytic function, a reductionof the lymphocyte response to polyclonal activators and an alteredfunction of B cells (D. Berger, Journal of Endotoxin Research, Vol. 4No. 1 (1997). This phase is characterized by an immunologicaldysfunction of the patient. The pneumonia produced by thecytomegalovirus (CMV) is more important in persons having an immunedeficiency, such as patients treated with immuno-suppressors as a resultof an organ transplant or a malignant process, as well as persons withnatural immunologic deficiencies such as hypogammaglobulinemia and withweakening chronical processes, for which reason a pneumonia produced byCMV may be a complication in any patient with a decreased immunologicalcapacity. Pneumonias of viral origin, do not respond to antimicrobialtreatment. The pneumonia produced by the grippe virus generally affectselderly persons that are immunologically depressed due to underlyingnon-infectious diseases, as for example, lung, cardiovascular or kidneydiseases. In all these cases it would be very useful to count on anagent with antiviral and/or immune system stimulating properties.

This invention is particularly important in the medical field. Theeffectiveness of the peptide disclosed herein is related to theantiviral and antibacterial effects, furthermore to activate the immunesystem and protect the host against bacterial or viral infectious. Thus,the peptide₃₁₋₅₂ (SEQ ID NO: 1) from Limulus anti-LPS factor is usefulin the treatment and/or prophylaxis of both viral and bacterialinfectious diseases, patients who are at a heightened risk of infectiondue to imminent surgery, injury, illness, or other such condition whichdeleteriously affects the immune system. The peptide is also useful inthe treatment of immumosuppressed patients. Take together, the peptideof this invention can be considered as a new immunomodulatory agent ofthe immune system with a broad spectrum for the treatment of infectiousdiseases.

DETAILED DESCRIPTION OF INVENTION

The invention relates to a peptide comprising the amino acids₃₁₋₅₂ (SEQID NO: 1) from Limulus anti-LPS factor protein isolate from a horseshoecrab such as Limulus polyphemus which enhance a host's immune defensemechanisms to infection but not induce an inflammatory response.

This peptide has been shown to have an antiviral effect to protect thecells against the viral infection in vitro assay. Furthermore,supernatant from human mononuclear cells incubated with the peptidecontain certain soluble mediator(s), such as IFN-γ, able to conferprotection to the cells Hep-2 against future viral infections. Anotherhand, this peptide protects against a direct infection in vivo. Theseadvantageous properties make the peptide of this invention useful in theprevention and treatment of infections because they selectively activateonly those components of the immune system responsible for the initialresponse to infection, without stimulating the release of certainbiochemical mediators that can cause adverse side effects. The peptidealso lacks the toxicity common to many immunomodulators.

The peptide of this invention is synthesized using a solid phaseprocedure. Crude peptide was extracted with a 30% acetic acid solutionin water, lyophilized and then purified by RP-HPLC. Molecular mass ofpurified peptide was verified using a JEOL JMS-HX110HF two sector massspectrometer equipped with a FAB gun. The resulting preparation isnon-antigenic, non-pyrogenic and is pharmaceutically acceptable foradministration to animals and humans.

The peptide comprising the amino acids31-52 (SEQ ID NO: 1) from Limulusanti-LPS factor protein of this invention can be used as safe,effective, therapeutic and/or prophylactic agents, either alone or asadjuvants, to enhance the immune response in humans and animals. Thepeptide comprising the amino acids sequences disclosed hereinselectively activate only those components that are responsible for theinitial response to infection caused both bacterial or viral pathogens,without stimulating or priming the immune system to release certainbiochemical mediators (e.g., TNF, IL-1) that can cause adverse sideeffects. As such, the present peptide can be used to prevent or treatinfectious diseases caused both bacterial or viral pathogens inmalnourished patients, patients undergoing surgery and bone marrowtransplants, patients undergoing chemotherapy or radiotherapy,neutropenic patients, HIV-infected patients, trauma patients, bumpatients, patients with chronic or resistant infections, all of who mayhave weakened immune systems. An immunocompromised individual isgenerally defined as a person who exhibits an attenuated or reducedability to mount a normal cellular and/or humoral defense to challengeby infectious agents, e.g., viruses, bacteria, fungi and protozoa. Aprotein malnourished individual is generally defined as a person who hasa serum albumin level of less than about 3.2 grams per deciliter (g/dl)and/or unintentional weight loss of greater than 10% of usual bodyweight.

More particularly, the method of the invention can be used totherapeutically or prophylactically treat animals or human who are at aheightened risk of infection due to imminent surgery, injury, illness,radiation or chemotherapy, or other such condition which deleteriouslyaffects the immune system. The method is useful to treat patients whohave a disease or disorder which causes the normal metabolic immuneresponse to be reduced or depressed, such as HIV infection (AIDS). Forexample, the method can be used to pre-initiate the metabolic immuneresponse in patients who are undergoing chemotherapy or radiationtherapy, or who are at a heightened risk for developing secondaryinfections or post-operative complications because of a diseases,disorder or treatment resulting in a reduced ability to mobilize thebody's normal metabolic responses to infection. Treatment with thepeptide has been shown to be particularly effective in mobilizing thehost's normal immune defenses, thereby engendering a measure ofprotection from infection in the treated host. It may be used in elderlypersons, immunologically depressed by non infectious diseases ascardiovascular or chronical diseases. It is known that these persons aresusceptible to viral pneumonia e.g. the grippe virus. It may also beeffective in the treatment of pneumonia due cytomegalovirus (CMV), whichare more important every day in persons having immunity deficiency, e.g.persons treated with immuno-suppressors as a result of an organtransplant. The treatment with this peptide could be particularlyattractive in strengthening the immune response in patients with traumaor it may be administered during the so called post-operational acutephase, where viral infections are generally associated producing severeconsequences, where the patients do not respond to the antimicrobialtreatments. Both in persons with traumas, as during the acutepost-operational phase, there is a deterioration of the immune responsegiven mainly by a decrease of the activity of the macrophages and theirinability to respond adequately during an infection (Berger et al.,Clin. Chem. Acta. 1995, Vol. 239: 121-130. Therefore, the agents thatstimulate the function of the macrophages, as the main mediators of theimmune response, would be of great therapeutic and prophylactic value ininfectious diseases of viral or bacterial origin. The treatment with thepeptide has demonstrated that it is particularly effective in mobilizingthe normal immune defenses of the host, producing an increase in theprotection of the host to the infection.

In another embodiment the peptide of the invention can be administrateto patients' undergoing “immunoparalysis” (Randow et al. , J. Exp. Med.1995, Vol. 181: 1887-1892.), defined as the reduction of the monocyticMHC class II, impaired monocytic antigen-presenting activity and theirdiminished ability to produce inflammatory cytokines. This phenomenon iscalled Compensatory Anti-inflammatory Responses Syndrome (CARS). Duringthe late hypoinflammatory phase in sepsis monocytic stimulation isuseful because restitute the deficient phenotype and function ofmonocytes from patients with “immunoparalysis”.

The present peptide is generally administered to an animal or human inan amount sufficient to produce immune system enhancement. The mode ofadministration of the peptide can be oral, enteral, parenteral,intravenous, subcutaneous, intraperitoneal, or intramuscular. The formin which the composition will be administered (e.g., tablet, capsule,solution, emulsion) will depend upon the route by which it isadministered. The quantity of the composition to be administered will bedetermined on an individual basis, and will be based at least in part onconsideration of the severity of infection or injury in the patient, thepatient's conditions or overall health, the patient's weight and thetime available before surgery, chemotherapy or other high-risktreatment. In general, a single dose will preferably containapproximately 5 mg to 10 mg of peptide per kilogram of body weight. Ingeneral, the composition of the present invention can be administratedto an individual periodically as necessary to stimulate the individual'simmune response.

An individual skilled in the medical arts will be able to determined thelength of time during which the composition is administrated and thedosage, depending upon the physical condition of the patient and thedisease or disorder being treated. As stated above, the composition mayalso be used as a preventative treatment to pre-initiate the normalmetabolic defenses, which the body mobilizes against infections.

The peptide can be used for the prevention and treatment of infectionscaused by a broad spectrum of bacterial, fungal, viral and protozoanpathogens. The prophylactic administration of peptide to a personundergoing surgery, either preoperatively, intraoperatively and/orpost-operatively, will reduce the incidence and severity ofpost-operative infections in both normal and high-risk patients. Forexample, in patients undergoing surgical procedures that are classifiedas contaminated or potentially contaminated (e.g., gastrointestinalsurgery, hysterectomy, cesarean section, transurethral prostatectomy)and in patients in whom infection at the operative site would present aserious risk (e.g., prosthetic arthroplasty, cardiovascular surgery),concurrent initial therapy with an appropriate antibacterial agent andthe present peptide preparation will reduce the incidence and severityof infectious complications.

In patients who are immunosuppressed, not only by disease (e.g., cancer,AIDS) but by courses of chemotherapy and/or radiotherapy, theprophylactic administration of the peptide preparation will be reducethe incidence of infections caused by a broad spectrum of opportunisticpathogens including many unusual bacteria, fungi and viruses.

In high risk patients (e.g., over age 65, diabetics, patients havingcancer, malnutrition, renal disease, emphysema, dehydration, restrictedmobility, etc.) hospitalization frequently is associated with a highincidence of serious nosocomial infection. Treatment with this peptidepreparation may be started empirically before characterization, use ofrespirators, drainage tubes, intensive care units, prolongedhospitalizations, etc. to help prevent the infections that are commonlyassociated with these procedures. Concurrent therapy with antimicrobialagents and the peptide preparation is indicated for the treatment ofchronic, severe, refractory, complex and difficult to treat infections.

The compositions administered in the method of the present invention canoptionally include other components, in addition to the peptide. Theother components that can be included in a particular composition aredetermined primarily by the manner in which the composition is to beadministered. For example, a composition to be administered orally intablet form can include, in addition to peptide, a filler (e g.,lactose) a binder (e.g., carboxymethyl cellulose, gum arabic, gelatin),an adjuvant, a flavoring agent, a coloring agent and a coating material(e.g., wax or plasticizer). A composition to be administered in liquidform can include peptide and, optionally, an emulsifying agent, aflavoring agent and/or a coloring agent. A composition for parenteraladministration can be mixed, dissolved or emulsified in water, sterilesaline, PBS, dextrose or other biologically acceptable carrier. Acomposition for topical administration can be formulated into a gel,ointment, lotion, cream or other form in which the composition iscapable of coating the side to be treated, e.g., wound site.

Compositions comprising peptide preparation can be administeredtopically to a wound site to stimulated and enhance wound healing andrepair. Wounds due to ulcers, acne, viral infections, fungal infectionsor periodontal disease, among other, can be treated according to themethods of this invention to accelerate the healing process.Alternatively, peptide preparation can be injected into wound orafflicted area. In addition to wound repair, the composition can be usedto treat infection associated therewith or the causative agents thatresult in the wound. A composition for topical administration can beformulated into a gel, ointment, lotion, cream or other form in whichthe composition is capable of coating the site to be treated, e.g.,wound site. A typical dosage for wound will be from about 5 mg to about10 mg.

In another embodiment this invention relates to hybrid polypeptidescontaining the mentioned peptide sequences wherein the preferred peptidesequences constitutes the N-terminus or the C-termninus of largerpolypeptide chains in a way that maintain its ability to enhance theimmune response and confers this ability to the hybrid polypeptide. Onepreferred hybrid polypeptide comprises a fusion of any of the preferredpeptides and heavy chain regions of IgG.

This invention also relates to scaffold proteins that properly exposedof the mentioned peptide sequences in such a way that maintains orenhances their ability to enhance the immune response and confers thisability to the hybrid polypeptide. The term “scaffold proteins” as usedherein refers to hybrid polypeptides, which include within theirpolypeptide chain one or more of the selected LALF sequences in a such away that the inserted segment forms an exposed loop in the structure ofthe fused protein, or polypeptide.

Peptide half life in vivo and other pharmacological parameters could beimproved with hybrid and scaffold polypeptides and proteins includingthe preferred peptide sequence. Also DNA sequences encoding thepeptides, or encoding hybrid or scaffold proteins containing the peptidesequence could be inserted in proper vectors to be expressed in vivo fortherapeutic purposes along with appropriated carriers, diluents,adjuvant or stabilizing solutions or chemicals.

The novel immunological activity from Limulus anti-LPS factor peptidedisclosed herein enhances the non-specific defenses of mammalianmononuclear cells and significantly increases their ability to respondto an infectious challenge. In another hand, it does not result inincreased body temperatures (e.g., fever) as has been reported with manynon-specific stimulants of those defenses. This critical advantage ofpeptide preparation may lie in the natural profile of responses itmediates in white blood cells. It has been shown that the effect of thepresent invention selectively activates immune responses but does notdirectly stimulate or prime cytokine (e g., TNF) release frommononuclear cells, thus distinguishing from other immunostimulants.

The invention is below illustrated with specific examples, which arepresented with the aim of illustration, but do not limit the extent ofthe invention.

EXAMPLES Example 1 Antiviral Effect of Peptide on the Cell Line HEP-2

Determination of the antiviral activity was estimated by means of theinhibition of the cytopathic effect produced by a virus on cell lineHep-2, it is a common procedure for determine antiviral activity ofinterferons ((Famillietti et al., 1981, Methods in Enzymology, Vol. 78:387-396). For this purpose 96 well plastic culture dishes (Nunc,Denmark) were used. 1×10⁵ cell/well in MEM medium supplemented with 10%fetal calf serum were growth up to monolayer formation (24 h). The nextday 100 ul of serial dilution's of the samples containing quantities of14 and 28 μg/ml of different peptide from LPS-binding proteins: Limulusanti-LPS factor₃₁₋₅₂ (LALF), Lipopolysaccharide Binding Protein₈₆₋₉₈(LBP), 18 kDa Cationic Antimicrobial Protein₁₀₈₋₁₂₈ (CAP-18) andBactericidal Permeability Increasing Protein₈₆₋₉₉ (BPI), were incubatedwith the cells for 24 h. Then the cells were washed with saline solutionand infected with 10⁻⁷ pfu of Mengo virus, provide by Institute ofMolecular Biology, University of Zurich, which is multiplied in the cellline L929 murine fibroblasts. 18 h later degree of cell destruction wasmeasured by fixation and staining, generally with crystal violet, forreading of cytophatic effect was used a photometer UltramicroanalyticalSystem (SUMA). A computer program in Pascal UCSD language for themicrocomputer system was developed for all calculations and corrections(Ferrero et al., 1994, Biotecnologia Aplicada. Vol. 1:). In this caseserial dilution's of human γ-IFN 70 000 IU/ml (MRI, Freeze-driedReference Human Recombinant Gamma Interferon) was used as standard inorder to define unit of antiviral activity. For this reason theantiviral protection shown by the peptide is related to InternationalUnits of γ-IFN (Ul/ml). As a negative control were used differentpeptides from proteins that bind endotoxin. LPS at 10 ng/ml was used asa positively control because is well known that it induced theproduction of IFNs (Manthey et al., 1994, J. Immunology. Vol. 153:2653-2663). One unit of antiviral activity is define as the quantity ofIFN able to produce an 50% inhibition of the viral replication.

The results are shown in Table 1.

TABLE 1 Antiviral effect shown by different peptide from LPS-bindingproteins on the cell line Hep - 2 Antiviral effect IU/ml γ-IFN PeptideConc. μg/ml 1 2 3 Mean ± SD LALF 14 μg/ml 35 31.7 22 29.5 6.75 28 μg/ml28.7 27 31.4 28.3 3.21 LBP 14 μg/ml 13 10 12.5 11.8 1.60 28 μg/ml 10 9.39 9.4 0.51 BPI 14 μg/ml 8 8.7 10 8.9 0.25 28 μg/ml 10 10.4 12.4 10 1.84CAP-18 14 μg/ml 9 8.7 9.2 8.9 1 28 μg/ml 12.2 9 9 10.9 1.22 LPS 0.01μg/ml   20.7 30 31.2 27.3 5.74 Cells 8.3 9 11 9.4 1.40 alone

Table 1 shows that only the peptide LALF is able to induce an antiviralstate in the cells Hep-2 defined as International Units/ml of γ-IFN.There was no effect for the other peptides derive from LPS-bindingprotein.

Example 2 Antiviral Effect of Peptide on the Cell Line MDBK

Determination of the antiviral activity was estimate by means of theinhibition of the cytopathic effect produced by a virus on cell lineMDBK, essentially as is described in the example 1. The cells used inthis approach were MDBK a kidney normal bovine cells line [ATCC No.6071], which are infected with 10⁻⁷ pfu of bovine enterovirus.Increasing quantities of LALF peptide were used in order to inducedprotection in the-cells: 10 μM, 20 μM and 40 μM. In this case serialdilution's of human α-IFN 70 000 IU/ml (MRI, Freeze-dried ReferenceHuman Recombinant Alpha Interferon) was used as standard in order todefine unit of antiviral activity. One unit of antiviral activity isdefining as the quantity of IFN able to produce a 50% inhibition of theviral replication. The FIG. 5 shows that LALF peptide is able to induceprotection in the cell line MDBK in a doses dependent manner.

Example 3 Supernatant From Human Mononuclear Cells Stimulated WithPeptide Limulus Anti-LPS Factor₃₁₋₅₂ LALF) Induce Antiviral Effect onHEP-2 Cell Line

Venous blood was obtained from healthy volunteers and Ficoll-Hypaquecentrifugation, fractionated mononuclear cells. The mononuclear cellswere washed and resuspended in endotoxin-free RPMI-1640 culture mediumsupplemented with 10% fetal calf serum. Concentration of 2×10⁶ cells/mlwere aliquoted into 24-well microtiter plates (Nunc, Denmark). The cellswere then incubated with 14 and 28 μg/ml of different peptides: Limulusanti-LPS factor₃₁₋₅₂ (LALF), Lipopolysaccharide Binding Protein₈₆₋₉₉(LBP) and 18 kDa Cationic Antimicrobial Protein₁₀₈₋₁₂₈ (CAP-18), at 37°C. for 18 hours in 5% CO₂. The cells were centrifuged at 3000 rpm for 5min and then serial dilutions of supematants were assays for antiviralactivity using the procedures described in the example 2.

The results are summarized in Table 2. Only LALF peptide used asstimulant of human peripheral blood mononuclear cells induce them torelease certain mediator capable of conferring protection to the Hep-2cells against viral infections.

TABLE 2 Supernatant from human mononuclear cells stimulated with LALFpeptide is able to induce an antiviral state on Hep - 2 cell lineAntiviral effect IU/ml γ-IFN Peptide Conc. μg/ml 1 2 3 Mean ± SD LALF 14μg/ml 28 27.6 20.4 25.3 4.27 28 μg/ml 28.4 28 20 25.4 4.73 LBP 14 μg/ml7 7.4 9.8 8.0 1.51 28 μg/ml 7.3 9 7.4 7.9 0.95 CAP-18 14 μg/ml 7.8 8 118.93 1.79 28 μg/ml 8.2 9 9.6 8.93 0.70 LPS 0.01 μg/ml   23 20 28.8 23.94.47 monocytes 12.6 9.2 8 9.9 2.38 alone

Example 4 Lack of in Vitro Stimulation of TNFα From Human MononuclearCells Incubated With Peptide

Venous blood was obtained from healthy volunteers and Ficoll-Hypaquecentrifugation, fractionated mononuclear cells. The mononuclear cellswere washed and resuspended in endotoxic-free RPMI 1640 mediumsupplemented with 10% fetal calf serum. 30×10⁶ cells were aliquoted intoplates p-60 (Nunc, Denmark). The cells were incubated with 14 or 28μg/ml LALF at 37° C. for 18 hours in 5% CO₂ and then centrifuged at 3000rpm during 5 min. The synthesis of TNFα in the supernatant wasdetermined by specific enzyme-linked immunoadsorbent assay (ELISA),kindly provided by Dr. W. Buurman (University Hospital Maastrich, TheNetherlands). The results are summarized in Table 3. LALF peptide usedas a stimulant at doses of 14-28 μg/ml alone did not induce increasedlevels of TNF-α over the control buffer treated cells.

TABLE 3 TNF - α synthesis by human mononuclear cells stimulated withvarious peptides from LPS-binding proteins Peptide Conc. μg/ml TNF - α(ng/ml)¹ LALF 14 μg/ml 0.84 28 μg/ml 0.80 LBP 14 μg/ml 0.80 28 μg/ml0.78 CAP-18 14 μg/ml 0.76 28 μg/ml 0.75 LPS 0.01 μg/ml   2.20 monocytes0.75 only ¹The values are the mean of three donors.

Example 5 In Vivo Protection Against Infection in Mice

A model of fulminating Gram-negative peritoneal sepsis was developed inmice BalB/c to characterize the efficacy of LALF peptide in protectingan immunologically intact host against serious infections. Groups ofmice received LALF peptide (14 μg/0.1 ml) or saline control (0.1 ml)intraperitoneally 20 hours prior to infectious challenge. A doses ofPseudomona aerukinosa (2×10⁸ bacterial/mice) producing a total mortalityof approximately 90% (Browder et al., 1987, Surgery. Vol. 102: 206-214)was used in order to induce an experimental peritonitis. Survivor's micewere counted each 24 hours during 120 hours after bacterial challenge.Survival data were analyzed by statistical test Kaplan-Meyer, thesignificance analysis was determined using a long-rank test, table 5.Blood was collected for TNF quantification from the retroorbital sinusat the time 0, 20, 40, 60, 90, 120, 180, 240 min in the control groupand peptide treated-mice. The concentration of TNF-α in mouse serum wasdetermined using a specific ELISA kindly provided by. Dr. W. Buurman(University Hospital Maastrich, The Netherlands). FIG. 4.

TABLE 4 Prophylactic effect of LALF peptide on survival in a mice modelof Gram - negative peritoneal sepsis Group Survival (%) P vs. SalineSaline Sol.  8% LALF peptide 36% P < 0.0098 prophylac.

The peptide protects mice from lethal bacterial challenge when it isadministrated prophylactically. The observed protection is attribute toa regulatory activity of peptide on the immune response of the host.This effect induced a decrease of the proinflammatory cytokine TNF FIG.4. This capacity may attenuate the lethal effect observed during thesepsis as a consequent of the systemic proinflammatory mediatorreleases.

Example 6 Lalf Peptide is Not Toxic and Does Not Affect the Levels ofTNF-α in Vivo

This approach was developed in mice BalB/c between 6-8 old. The micewere injected intravenously with 50 μg of peptide in 0.1 ml [2.5 mg/kgof body weight] or saline solution as a control. The behavior of animalswas observed each 4 hours during 24 h. Survival was followed for oneweek. Blood was collected for TNF-α quantification from the retroorbitalsinus at the time 0 h. 6 h, 24 h and 48 h in the control group andpeptide treated-mice. The concentration of TNF-α in mouse serum wasdetermined using a specific ELISA kindly provided by Dr. W. Buurman(University Hospital Maastrich, The Netherlands). All mice [10animals/group] survivor beyond of one week. LALF peptide is not toxic invivo and does not stimulate TNF-α production, FIG. 3.

Example 7 Regulation Effect of Lalf-derived Peptide on Immune Responsein activated-peritoneal Macrophages

Peritoneal elicited cells were obtained from Balb/c mice (CENPALAB,Havana, Cuba). Primary mouse macrophages was collected from theperitoneal cavity 4 days after intraperitoneal injection with 1 ml of 4%Brewer's thioglicollate broth. Cell were maintained in RPMI 1640supplemented with 10% heat-inactivated fetal bovine serum, 2 mML-glutamine, 200 units/ml penicillin, and 200 μg/ml streptomycin at 37°C. in 5% CO₂/96% air.

Assay for Secretion of NO₂-(Jin, F²⁹, Nathan, C, Radzioch, 0, and Ding,A. Cell, 1997, 88: 417-426). Cells were plated in 96-well plates at2×10⁶ cells /ml in 200 μl of medium and incubated at 37° C. for 2 h,then non-adherent cells were removed by repeated washing with PBS.Macrophage were pre-incubated with 5 μM of LALF peptide during 18 h,afterward were washed with RPMI medium in order to eliminated thepeptide. Then the macrophages were treated 72 hr with LPS of E. coli0111:B4, Sigma (1 μg/ml). Cell viability was assessed bymitochondrial-dependent reduction of MTT to formazan (Szabo³⁰, c,Mitchell, J A, Gross, S S, Thiemermann, C, and Vane, JR. J. Pharm. Exp.Therap., 1992, 265: 674-80). 50 μl of cultured medium was mixed withequal volume of Greiss's reagent (1% sulfanilamide, 0.1%naphthylethylenediamine dihydrochloride, 2.5% H₃PO₄). Absorbance at 540nm was recorded in a microplate reader (SUMA) with sodium nitrite asstandards (Green²⁸, L C, Wagner, D A, Glogowski, J, et al. Anal Biochem1982; 126: 131-8). Nitrite content of similarly incubated cell-freemedium was subtracted.

TNF-α assay: Cells were plated in 96-well plates at 2×10⁶ cells/ml in200 μl of medium and incubated at 37° C. for 2 h. Non-adherent cellswere removed by repeated washing with PBS. Macrophages werepre-incubated with 5 μM of LALF peptide during 18 h, afterward werewashed with RPMI medium in order to eliminate the peptide. Thenmacrophages were treated with LPS of E. coli 0111:B4, Sigma (1 μg/ml).TNF-α was determined in supematants from cultured cells at the time 1 h,2 h, 4 h, 6 h and 24 h used a kit of ELISA kindly provided by [Dr. W.Buurman Univ. Hosp. Maastrich, Netherlands].

As shows the FIG. 6 the TNF levels were down regulated inmacrophages-treated peptide. On contrary NO production was up regulated,FIG. 7. The viability of the cells did not affect during the assay. Theviability was never found lower than 98%.

It seems to be a preferring and specifically modulation of LPS-inducedresponse. Take together, may be the peptide mediate some kindinteraction with the immune system cells and lead to immunoregulatoryeffect able to enhance and/or modulate the immune response in the host.

EXAMPLE 8 Stimulation of PBMCs With LALf-derived Peptide. Analysis ofCytokine Gene Expression Using The Ribonuclease Protection Assay (RPA)

The Ribonuclease Protection Assay (RPA) is a highly sensitive andspecific method for the detection, quantification and characterizationof RNA molecules (Gilman³², M. In Current Protocols in MolecularBiology. J. Wiley and Sons, New York, p. 4.7.1-7.7.8. 1988 and Dent³³,A. L., Shaffer, A. L., Yu X., Allman, D., Staudt, L. M., 1997. Cytokineexpression and germinal center formation by Bcl-2. Science. 276:589-592). Samples of total RNA from stimulated and nonstimulated humanPBMCs were analyzed for distinct mRNA species by PharMingen'sRiboQuant™. Multi-Probe Ribonuclease Protection Assay System with thehck-1 probe template set (Cat. No. 45031P) which including mRNA speciesanalyses for a different cytokine eg. (IL-5, IL-4, IL-10, IL-15, IL-9,IL-2, IL-13, IFN-γ).

Briefly, a high specific activity, [³²P]-labeled anti-sense cytokine RNAprobe set [hck-1, Cat.No.45031P] is hybridized in excess to samplestarget RNA from PBMCs incubated with 5 μM of the peptide for 18 hours.Free probe and other single stranded RNA molecules are digested withRnases, whereas annealed probe: target RNA duplexes are protected fromRNase digestion. These remaining Rnase-protected probes are purified,resolved on denaturing polyacrylamide gels according to their size, andimaged by autoradiography. The identity and quantity of each mRNAspecies in the original RNA sample can then be determined based on thesignal intensities given by the appropriately sized of protected probefragment bands. Unprotected probe set and a probe for housekeeping genetranscripts are incorporating in order to identify and quantify the RNAmolecules in the original samples. For furthermore specificities seeHot-Lines (PharMingen) Vol.3, No.1 pp.1 1997 or PharMingen RiboQuant™Multi-Probe Rnase Protection Assay System, Instruction Manual 4^(th)Edition, August 1997.

Total RNA was obtained using a Single-Step Method of RNA isolation byAGPC Extraction (Chomczynski³⁴ et al. 1987, Analytical Biochemistry.Vol. 162: 156-159).

As shows the FIG. 8 the peptide is able to preferentially induce IFN-γ,IL-2 and IL-13 gene expression in human PBMCs. Expression and subsequentrelease of IFN-γ might be the responsible of the antiviral propertyobserved in supernatants from PBMCs incubated with the peptide.Furthermore, this typically Th-1 response characterized by production ofIL-2 and IFN-γ may correlated with the protect effect of the peptideduring P. aeruginosa infection in mice. Because, has been reported thatIFN-γ decreases the inflammatory response in chronic P. aeruginosainfection in rats (H. K. Johansen³⁵, H. P. Hougen, J. Rygaard and N.Hoiby. Clin. Exp. Immnunol. 1996, 103: 212-218.). Besides, have beenfound that treatment with IFN-.gamma. influence the cell-mediatedresponse and therapeutic potential has been demonstrated in vivo againstCryptococcus neoformans (Joly³⁶ V, Saint-Julien L. Carbon C, Yeni P.Journal of Infect. Dis. 1994; 170: 1331-4), Toxoplasma gondii(Benedetto³⁷ N, Auriault C, Darcy F et al. Eur Cytokine Net. 1991; 2:107-14) and Klebsiella pneumoniae (Hagen³⁸ TLM, Vianen von W,Bakker-Woudenberg IAJM. J. Infect Dis. 1995; 171: 385-92). Here weshowed that LALF-derived peptide is able to produce immune systemenhancement and can be used to prevent or treat infectious diseasescaused by a broad spectrum of pathogens. Furthermore, the LALF-derivedpeptide can be used in patients who may have a weakened immune system.Statistical methods. All values in the figures and table are expressedas mean±SD. Statistical analysis was done by using the Kruskal Wallistest. The different between groups was determined by a Dunn test. A pvalues less than 0.05 was assumed to be significant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows the sequence of the peptide LALF (SEQ ID NO: 1) that isuse to define the activity disclosed herein.

FIG. 2: Shows RP-HPLC chromatographic purification of the LALF peptide.Peak A: elution of the peptide with 98% of purity.

FIG. 3: Shows the change in serum TNF levels, over time, taken from miceintravenously infused with saline solution or 50 μg of LALF peptide.

FIG. 4: Comparison of systemic levels of TNF in LALF-derived peptidetreated-mice and control group. Eight-week-old mice were injected i.p.with 14 μg of peptide in 0.1 ml saline or saline alone. Twenty hoursafter the mice were injected i.p. with a lethal doses of P. aeruginosa.At 0, 20, 40, 60, 90, 120, 180 and 240 min following the challenge, themice were bled from the retroorbital sinus and the serum concentrationof TNF was measured.

FIG. 5: Shows the antiviral properties of LALF peptide on the cell lineMDBK. The cells were treated with increasing quantities of peptideduring 18 hours. Afterwards, the cells were challenged with a bovineenterovirus. Antiviral activity is referred as U/ml α-IFN.

FIG. 6: Regulatory effect of LALF derived-peptide on TNF-α production instimulated peritoneal macrophages. The macrophages were pretreated with5 μM of LALF peptide during 18 hours and then the cells were activatedwith 1 μg of E. coli LPS. TNF-α was measured in the cells culturedsupernatants at different time.

FIG. 7: Regulatory effect of LALF derived-peptide on NO production instimulated peritoneal macrophages. The macrophages were pretreated with5 μM of LALF peptide during 18 hours and then the cells were activatedwith 1 μg of E. coli LPS during 72 h. Levels of NO were measured in thecells cultured supernatants using the Greiss method.

FIG 8: Samples of total RNA from stimulated human PBMCs with 5 μM ofpeptide for 18 hours were analyzed for distinct mRNA species by usingPhaiMingens RiboQuant Multi-Probe Ribonuclease Protection Assay Systemwith the hck-1 probe template set (Cat. No. 45031P). The autoradiogramfrom this analysis shows the hck-1 as an unprotected probe set (Lane 1).Also shows the corresponding RNase-protected probes followinghybridization with: total RNA isolated from PBMCs nonstimulated (Lane 2)and total RNA isolated from PBMCs stimulated with 5 μM of LALF peptideduring 18 hours (Lane 3).

REFERENCES

1. Morrison and Ulevitch. Am. J. Pathol. Vol. 93: 527 (1978).

2. Bone., Annals Int. Med. Vol. 115: 457 (1991).

3. Helene et al. , Infection and Immunity. Vol. 62: 1185 (1994).

4. Schumann et al., Science, Vol. 249: 1429 (1990).

5. Wright et al., J. Exp. Med. Vol. 170: 1231 (1989).

6. Wright et al., Science. Vol. 249: 1431 (1990).

7. Wright et al. , J. Exp. Med. Vol. 173: 1281 (1991).

8. Worthen et al. J. Clin. Investig. Vol. 20: 2526 (1992).

9. Gazzano-Santoro., WO 95/00641.

10. Wainwright et al., WO 92/20715.

11. Warren et al. Infect. Immun. Vol. 60: 2506-2513 (1992).

12. Hoess et al., WO 95/05393.

13. Hoess et al., The Embo Journal. Vol.12: 3351-3356 (1993).

14. Randow et al., J. Exp. Med. Vol. 181: 1887-1892 (1995).

15. Famillietti et al. Methods in Enzymology. Vol. 78: 387-396 (1981).

16. Ferrero et al., Biotecrnologia Aplicada. Vol. 1 (1991).

17. Sheila et al. Infect and Immunity. Vol. 63; 601-608 (1995).

18. Browder et al. Surgery. Vol. 102: 206-214 (1987).

19. Berger et al, Journal of Endotoxin Research. Vol. 4: 17-24 (1997).

20. Berger et al., Clin. Chim. Acta. Vol. 239: 121-130 (1995).

21. Capodici C., Weiss J. Journal of lmmnunology. Vol.156 (12): 4789-96(1996).

22. Taylor A H., Heavner G., Nedelman M. et al., J. Biol. Chem. Vol. 270(30): 17934-8 (1995).

23. Garcia C., Saladino R., Thompson C. et al., Crit. Care. Med. Vol. 22(8): 1211-8 (1994).

24. Battafaraono R. J. , Dahlberg P S., Ratz C. A. et al., Surgery Vol.118 (2): 31824 (1995).

25. Fletcher M. A., Kloczewiak M. A., Loiselle P. M. et al., J. Infect.Dis. Vol. 175 (3): 621-32 (1997).

26. Ried C., Wahl C., Miethke T. et al. J. Biol. Chem. Vol. 271 (45):28120-7 (1996).

27. Kloczewlak M., Black K. M., Loiselle P. et al., J. Infect. Dis. Vol.170 (6): 1490-7 (1994).

28. Green L C. Wagner D A., Glogowski, J. et al; Anal Biochem. Vol. 126:131-8 (1982).

29. Jin, F, Nathan, C, Radzioch, D, and Ding A. Cell Vol. 88: 417-426(1997).

30. Szabo, c, Mitchell, J A, Gross, S S, Thiemermann, C, and Vane, J R.J. Pharm. Ex,. Therap., 1992, 265: 67480)

31. Roth R I. Su D H. Child A H. Wainwright N R and Levin J. Journal ofInfectious Diseases. Vol. 177 (2): 388-394, 1998.

32. Gilman, M. In Current Protocols in Molecular Biology. J. Wiley andSons, New York, p. 4.7.1-4.7.8. 1988.

33. Dent, A L., Shaffer, A. L, Yu X., Allman, D, Staudt, L. M., 1997.

34. Chomczynski et al. 1987, Analytical Biochemistry. Vol. 162; 156-159.

35. H. K. Johansen, H. P. Hougen, J. Rygaard and N. Hoiby. Clin. Exp.Immnunol. 1996, 103: 212-218.

36. Joly V, Saint-Julien L, Carbon C, Yeni P. Journal of Infect. Dis.1994; 170: 1331-4.

37. Benedetto N, Aunault C, Darcy F et al. Eur Cytokine Net. 1991; 2:107-14.

38. Hagen TLM, Vianen von W, Bakker-Woudenberg IAJM. J. Infect Dis.1995; 171: 385-92.

1 1 22 PRT Limulus polyphemus 1 Cys His Tyr Arg Ile Lys Pro Thr Phe ArgArg Leu Lys Trp Lys Tyr 1 5 10 15 Lys Gly Lys Phe Trp Cys 20

What is claimed is:
 1. A viral vaccine comprising a peptide or proteinhaving the amino acid residues 31-52 (SEQ. ID NO.1) of Limuluspolyphemus anti-LPS factor, wherein said peptide or protein is anadjuvant in said viral vaccine.